Comparisons of Physicochemical Properties, Bacterial Diversities, Isoflavone Profiles and Antioxidant Activities on Household and Commercial doenjang

In this study, the physicochemical properties (pH, acidity, salinity, and soluble protein), bacterial diversities, isoflavone contents, and antioxidant activities of doenjang (fermented soy paste), household doenjang (HDJ), and commercial doenjang (CDJ), were assessed and compared. The values of pH 5.14–5.94 and acidity 1.36–3.03%, indicated a similar level in all doenjang. The salinity was high in CDJ at 12.8–14.6%, and the protein contents (25.69–37.54 mg/g) were generally high in HDJ. Forty-three species were identified from the HDJ and CDJ. The main species were verified to be Bacillus amyloliquefaciens (B. amyloliquefaciens), B. amyloliquefaciens subsp. plantarum, Bacillus licheniformis, Bacillus sp. and Bacillus subtilis. Comparing the ratios of isoflavone types, the HDJ has an aglycone ratio of >80%, and 3HDJ indicates a ratio of isoflavone to aglycone of 100%. In the CDJ, except 4CDJ, glycosides account for a high proportion of more than 50%. The results of antioxidant activities and DNA protection effects were variedly confirmed regardless of HDJs and CDJs. Through these results, it is judged that HDJs have a variety of bacterial species compared to CDJs, and these are biologically active and converted from glycoside to aglycone. Bacterial distribution and isoflavone contents could be used as basic data.


Introduction
Soybeans have been used for the long term in East-Asian countries as one of the world's three most extensive food resources. They supplement nutrients in rice-centered diets, possessing a substantial amount of protein, essential fatty acids, organic acids, minerals, and vitamins generally lacking in grains. Some physiologically active substances, including isoflavone, polyphenol, flavonoid, dietary fiber, soy oligosaccharide, saponin, and lecithin are more greatly found in soybeans than in the other grains, which showed anti-aging, anticancer, anti-arteriosclerosis, and hypoglycemic effects [1][2][3]. The raw soybean materials enriched with antioxidant compounds are further converted into different functional metabolites after heating, germination, and fermentation [4,5]. Notably, the isoflavone aglycones, such as daidzein, glycitein, and genistein, were extracted from the isoflavone glycosides of soybean during fermentation [6]. Moreover, phenolic compounds such as vanillic acid, chlorogenic acid, p-coumaric acid, ferulic acid, caffeic acid, and saponins have also been reported from soybeans [7][8][9].

Physicochemical Properties of HDJs and CDJs
The differences in the physicochemical qualities of HDJ and CDJ are shown in Table 1. The pH value ranged from 5. 31-5.94 in HDJs, but 5.14-5.64 was obtained in CDJs. The acidity was also 1.36-3.03% in HDJs and 1.59-2.87% in CDJs. Among these, the 4HDJ demonstrated the highest acidity of 3.03%. There was no significant difference among these, and the highest pH was 5.94 in 4HDJ. Obviously, no significant differences in pHs and acidities were observed among the HDJ and CDJ samples. The average pH values of traditional household doenjang were higher than those of commercial doenjang [21,22], which was almost consistent with this study (average pH of HDJ and CDJ: pH 5.66 and 5.45). In fact, the acidity of traditional doenjang was higher than that of commercial doenjang samples [23]. In a previous study, during prolonged doenjang fermentation, organic acids and free fatty acid contents were escalated with lipolysis and amino acid biodegradation, but this has slightly deviated in this study.
In fact, 8.2-10.2% salinity was found in the HDJ samples, but a moderately higher salinity (12.8-14.6%) was recorded in the CDJ samples. The soluble protein contents were estimated at 37. 54, 35.37, 25.79, and 28.79 mg/g in the 1HDJ, 2HDJ, 3HDJ, and 4HDJ samples, respectively. Besides, it was estimated that 27.73, 20.53, 20.84, and 50.83 mg/g, were in the 1CDJ, 2CDJ, 3CDJ, and 4CDJ samples, respectively. To sum up, the soluble protein contents of the HDJ samples were decently higher than that of the CDJ samples. Jeon et al. [24] reported that the salinity value (11.77-14.22%) of traditional doenjang showed higher than that observed (10.8-11.4%) in commercial doenjang. While, several studies reported that traditionally manufactured doenjang showed higher salinity than the commercial (modified method) doenjang [23,25]. However, the salinity results were remarkably higher in all types of commercial doenjang samples compared with the traditional doenjang samples. Kim et al. [26] reported that the salinity of doenjang differs regardless of the manufacturing method, following as the 11 traditional doenjang showed 8.6-14.2% and the three commercial doenjang showed 10.8-15.8%. Most of the previous studies reported that the salinity of traditional doenjang showed higher salt contents, which is used to increase the storage periods [27]. Unlike with former reports about traditional doenjang salinity, the salinity was found to be lower in HDJs, which might be the following reasons: HDJs were separated into liquid (Kanjang) and solid (doenjang) during fermentation, followed by the removal of salt from doenjang with the liquid. The liquid-separated doenjang was mixed with steamed soybean or meju powder [28].

Comparison of Phenolic, Flavonoid, and Isoflavone Content on HDJs and CDJs
The total phenolic (TP), total flavonoid (TF), and total isoflavone contents of HDJ and CDJ samples are indicated in Figure 3., Table 3, and  16.08 (3CDJ), and 27.69 (4CDJ) GAE mg/g. The HDJs and CDJs did not significantly differ in total phenolic content ( Table 3). The TF contents were estimated 0.62, 0.89, 0.24, and 0.54 RE mg/g in 1HDJ, 2HDJ, 3HDJ, and 4HDJ samples, respectively. It is estimated 0.24, 0.58, 0.25, and 0.87 RE mg/g in 1CDJ, 2CDJ, 3CDJ, and 4CDJ, respectively. The TF content also did not exhibit a significant difference between HDJs and CDJs, like TP content. The total isoflavone contents were detected as 1460.80, 1314.22, 482.80, 899.38, 885.58, 1084.60, 993.84, and 1212.47 μg/g in 1HDJ, 2HDJ, 3HDJ, 4HDJ, 1CDJ, 2CDJ, 3CDJ, and 4CDJ samples, respectively. Total isoflavone content was not significantly different between HDJ and CDJ. Among the HDJ samples, two samples were confirmed to be higher than the CDJ samples, but one sample was confirmed to be the lowest among the HDJ and CDJ samples.
Glycoside, malonylglycoside, acetylglycoside, and aglycone isoflavone contents and ratio of HDJ and CDJ samples are indicated on Table 3 and Figure 4. Isoflavone glycosides

Comparison of Phenolic, Flavonoid, and Isoflavone Content on HDJs and CDJs
The total phenolic (TP), total flavonoid (TF), and total isoflavone contents of HDJ and CDJ samples are indicated in Figure 3, Table 3, and  16.08 (3CDJ), and 27.69 (4CDJ) GAE mg/g. The HDJs and CDJs did not significantly differ in total phenolic content ( Table 3). The TF contents were estimated 0.62, 0.89, 0.24, and 0.54 RE mg/g in 1HDJ, 2HDJ, 3HDJ, and 4HDJ samples, respectively. It is estimated 0.24, 0.58, 0.25, and 0.87 RE mg/g in 1CDJ, 2CDJ, 3CDJ, and 4CDJ, respectively. The TF content also did not exhibit a significant difference between HDJs and CDJs, like TP content. The total isoflavone contents were detected as 1460.80, 1314.22, 482.80, 899.38, 885.58, 1084.60, 993.84, and 1212.47 µg/g in 1HDJ, 2HDJ, 3HDJ, 4HDJ, 1CDJ, 2CDJ, 3CDJ, and 4CDJ samples, respectively. Total isoflavone content was not significantly different between HDJ and CDJ. Among the HDJ samples, two samples were confirmed to be higher than the CDJ samples, but one sample was confirmed to be the lowest among the HDJ and CDJ samples. contained only aglycones. The ratio of aglycones in HDJs was more than 80% (88.02, 81.32, 100, and 87.32%), as seen in Figure 4. In contrast, all types of isoflavones were found in CDJs, where glycosides had occupied the central portion of isoflavones in 1CDJ, 2CDJ, and 3CDJ, except in 4CDJ. Notably, the aglycone content was found to be 81.54% in the 4CDJ sample. The glycoside contents in 1CDJ, 2CDJ, 3CDJ, and 4CDJ were found as 450.34, 807.65, 599.94, and 105.47 μg/g, respectively, and the aglycone content was 266.43, 123.45, 265.99, and 988.60 μg/g, respectively. Consequently, glycoside content was highest in 2CDJ (807.65 μg/g), and aglycone content was highest in 4CDJ (988.60 μg/g). To sum up, the glycoside was highest in 2CDJ (74.46%), and aglycone was highest in 4CDJ (81.54%), the overall isoflavone ratio in CDJs. Figure 5 shows a typical HPLC chromatogram of 50% ethanol doenjang extracts. In the case of 3HDJ, only isoflavone aglycone forms, such as daidzein, glycitein, and genistein, were found.  Table 3. Comparison of glycoside, malonylglycoside, acetylglycoside, and aglycone isoflavone contents on household and commercial doenjang.  Glycoside, malonylglycoside, acetylglycoside, and aglycone isoflavone contents and ratio of HDJ and CDJ samples are indicated on Table 3 and Figure 4. Isoflavone glycosides and -malonylglycosides were found in 1HDJ (117.12 and 4.11 µg/g), 2HDJ (164.5080.97 µg/g), and 4HDJ (80.97 and 33.11 µg/g), except 3HDJ. Isoflavone-acetylglycosides were found only in 1HDJ (3.76 µg/g). Aglycone type isoflavone was the highest in all HDJ: 1285.81(1HDJ), 1068.75 (2HDJ), 482.80 (3HDJ), and 785.30 (4HDJ) µg/g. Notably, 3HDJ contained only aglycones. The ratio of aglycones in HDJs was more than 80% (88.02, 81.32, 100, and 87.32%), as seen in Figure 4. In contrast, all types of isoflavones were found in CDJs, where glycosides had occupied the central portion of isoflavones in 1CDJ, 2CDJ, and 3CDJ, except in 4CDJ. Notably, the aglycone content was found to be 81.54% in the 4CDJ sample. The glycoside contents in 1CDJ, 2CDJ, 3CDJ, and 4CDJ were found as 450.34, 807.65, 599.94, and 105.47 µg/g, respectively, and the aglycone content was 266.43, 123.45, 265.99, and 988.60 µg/g, respectively. Consequently, glycoside content was highest in 2CDJ (807.65 µg/g), and aglycone content was highest in 4CDJ (988.60 µg/g). To sum up, the glycoside was highest in 2CDJ (74.46%), and aglycone was highest in 4CDJ (81.54%), the overall isoflavone ratio in CDJs. Figure 5 shows a typical HPLC chromatogram of 50%   Shukla et al. [21] reported that no individual differences in TP content were observed in doenjang made with meju and commercial doenjang. Ahn et al. [34] reported that the TP content of traditional doenjang and commercial doenjang ranged from 18.71-25.78 GAE mg/mL. Most traditional doenjang is usually made using soybeans, salt, and water, whereas commercial doenjang components are koji, soybean, wheat, barley, salt, and water. Therefore, TP content among doenjang types differs according to doenjang composition material [17,18]. Kim et al. [25] reported that the major isoflavone of traditional doenjang was daidzein and genistein. Generally, isoflavones in soybean are found in glycosides form, e.g., genistin, daidzin, glycitin, which are converted into aglycone e.g., genistein, daidzein, glycitein by β-glucosidase activities of microorganisms [35][36][37]. In addition, Kwak et al. [38] reported that the aglycone contents increased with the progress of fermentation periods of doenjang. In fact, the household doenjang fermentation time was longer than the commercial doenjang. It is supposed that the high aglycone ratio and content in HDJs were because of the longer fermentation period, while the high glycoside ratio and content in CDJs were due to the short fermentation period. These results lead us to conclude that the distribution of glycosides and aglycone would be an indicator to estimate the approximate fermentation period of doenjang. Shukla et al. [21] reported that no individual differences in TP content were observed in doenjang made with meju and commercial doenjang. Ahn et al. [34] reported that the TP content of traditional doenjang and commercial doenjang ranged from 18.71-25.78 GAE mg/mL. Most traditional doenjang is usually made using soybeans, salt, and water, whereas commercial doenjang components are koji, soybean, wheat, barley, salt, and water. Therefore, TP content among doenjang types differs according to doenjang composition material [17,18]. Kim et al. [25] reported that the major isoflavone of traditional doenjang was daidzein and genistein. Generally, isoflavones in soybean are found in glycosides form, e.g., genistin, daidzin, glycitin, which are converted into aglycone e.g., genistein,

Comparison of Antioxidant Activities on HDJs and CDJs
The DPPH, ABTS, and hydroxyl radical scavenging activities and FRAP of HDJ and CDJ are shown in Figure 6. Interestingly, no significant differences in DPPH radical scavenging activities between HDJ and CDJ samples were found. In the case of HDJ, the highest DPPH radical scavenging activity was found in 2HDJ samples while it was obtained maximum in 1CDJ among other CDJ samples ( Figure 6A). Notably, at 100, 250, and 500 µg/mL of sample concentrations, the 2HDJ showed maximum 29.23%, 48.46%, and 86.92% DPPH radical scavenging activities, respectively. Similarly, the 1CDJ demonstrated the highest 29.68%, 49.36%, and 88.73% DPPH activities, respectively. Among these doenjang samples, the highest DPPH activities were provided by the 1CDJ samples in this study ( Figure 6A). scavenging activity and FRAP were significantly connected with aglycone and phenolic compounds. However, Shukla et al. [21] reported that doenjang's FRAP activity was slightly crosslinked with phenolic compounds, which agreed with our study. Subsequently, aglycones, phenolics, and flavonoids influenced antioxidant activities without showing any consistent patterns. Previous studies reported that isoflavone aglycone and phenolic compound content influenced antioxidant activity [7,45,46]. However, phenolics, flavonoids, and isoflavones did not consistently demonstrate a pattern with antioxidant activity, like DPPH, ABTS, FRAP reducing power, and hydroxyl radical scavenging activities between HDJs and CDJs. However, when they were compared and analyzed comprehensively, samples with high phytochemicals demonstrated high antioxidant activity.

Comparison of DNA Damage Protecting Activity on HDJs and CDJs
The DNA damage protecting activity of HDJs and CDJs against oxidative stress (Fenton's reagent) is shown in Figure 7. In this study, Fenton's reagent was used as an oxidizing agent to verify the OC (Open Circular DNA), LIN (Linear DNA), and SC (Supercoil DNA) protecting activity of HDJs and CDJ samples. The treatment of Fenton's reagent to plasmid DNA showed OC, LIN, and SC at about 8 kb, 3 kb, and 2 kb, respectively, in the untreated positive control. In the case of the negative control, Fenton's reagent treatment caused the damage to all three DNAs damaged. Consequently, the DNA could not be identified as shown in line 4 ( Figure 7). All OC, LIN, and SC DNA were defined in 1HDJ, 2HDJ, 3HDJ, and 4HDJ, but 1HDJ and 4HDJ demonstrated the strongest OC DNA band, followed by LIN and SC. Besides, 2HDJ and 3HDJ demonstrated the DNA protective effect of the same band pattern as the positive control. DNA protecting activity of 1CDJ, 2CDJ, 3CDJ, and 4CDJ also verified the OC, LIN, and SC DNA and household doenjang. Among them, 1CDJ demonstrated the strongest OC DNA band, followed by LIN and SC, and 2CDJ, 3CDJ, and 4CDJ exhibited the same DNA protective effect as a positive control.
Boukhris et al. [47] reported that flower extract caused no damage to DNA. Still, the bands' fluorescence intensity was altered because the bands of supercoil DNA weakened, Additionally, the 2HDJ and 1CDJ showed the highest ABTS radical scavenging activities among the HDJ and CDJ samples, respectively ( Figure 6B). At 50, 100, and 250 µg/mL sample concentration, the 2HDJ showed the highest 29.99%, 51.97%, and 95.95% among others HDJ, while 1CDJ showed maximum 30.49%, 27.14%, and 97.94%, ABTS radical scavenging activities, respectively. However, the 3HDJ showed the lowest, and 1CDJ showed the highest ABTS radical scavenging activity among all other doenjang samples ( Figure 6B). Not many significant differences in hydroxyl radical scavenging activities between the HDJ and CDJ samples were observed ( Figure 6C), as it was pronounced for DPPH and ABTS radical scavenging activities. In fact, the highest hydroxyl radical scavenging activity was expressed by the 2HDJ and 2CDJ samples, among other HDJ and CDJ, respectively. At 100, 250, and 500 µg/mL of sample concentration, the 2HDJ showed 20.26%, 34.51%, and 59.02%, whereas 1CDJ expressed 21.31%, 36.61%, and 63.22%, hydroxyl radical scavenging activities, respectively. At 500 µg/mL sample, the lowest activity obtained 28.97% in 3HDJ, whereas the highest activity was 63.22% in 2CDJ ( Figure 6C).
The fermentation process increased the antioxidant activity of fermented soybean foods, such as tofu, doenjang, and soymilk [39,40]. Generally, the fermentation period of household doenjang was longer than that of the commercial doenjang [18], but the radical scavenging activities of the HDJs was not significantly greater than that of the CDJs. It is judged to occur due to the decrease in phenolic contents in the manufacturing environment of HDJs compared to the CDJs. Similar concentrations of the samples showed higher activity in ABTS than in DPPH. This was observed because DPPH reacts with free radicals, but ABTS reacts with different free radicals, including peroxyl, hydroxyl, alkoxyl, and inorganic radicals, to form stable ABTS + type cationic radicals; due to having these differences, a difference in binding capacity to antioxidants occurred [41]. Therefore, ABTS + measured the antioxidant power of hydrophilic and hydrophobic substances, so it was judged to exhibit higher scavenging activity compared to DPPH [42]. The hydroxyl radical scavenging activity of fermented soybean yogurt and fermented soybean solid was estimated at 39% (500 µg/mL) and 49% (200 µg/mL) [43,44], which indicated a higher amount compared to doenjang used in the study. According to the report by Lee et al. [7], radical scavenging activity and FRAP were significantly connected with aglycone and phenolic compounds. However, Shukla et al. [21] reported that doenjang's FRAP activity was slightly crosslinked with phenolic compounds, which agreed with our study. Subsequently, aglycones, phenolics, and flavonoids influenced antioxidant activities without showing any consistent patterns. Previous studies reported that isoflavone aglycone and phenolic compound content influenced antioxidant activity [7,45,46]. However, phenolics, flavonoids, and isoflavones did not consistently demonstrate a pattern with antioxidant activity, like DPPH, ABTS, FRAP reducing power, and hydroxyl radical scavenging activities between HDJs and CDJs. However, when they were compared and analyzed comprehensively, samples with high phytochemicals demonstrated high antioxidant activity.

Comparison of DNA Damage Protecting Activity on HDJs and CDJs
The DNA damage protecting activity of HDJs and CDJs against oxidative stress (Fenton's reagent) is shown in Figure 7. In this study, Fenton's reagent was used as an oxidizing agent to verify the OC (Open Circular DNA), LIN (Linear DNA), and SC (Supercoil DNA) protecting activity of HDJs and CDJ samples. The treatment of Fenton's reagent to plasmid DNA showed OC, LIN, and SC at about 8 kb, 3 kb, and 2 kb, respectively, in the untreated positive control. In the case of the negative control, Fenton's reagent treatment caused the damage to all three DNAs damaged. Consequently, the DNA could not be identified as shown in line 4 ( Figure 7). All OC, LIN, and SC DNA were defined in 1HDJ, 2HDJ, 3HDJ, and 4HDJ, but 1HDJ and 4HDJ demonstrated the strongest OC DNA band, followed by LIN and SC. Besides, 2HDJ and 3HDJ demonstrated the DNA protective effect of the same band pattern as the positive control. DNA protecting activity of 1CDJ, 2CDJ, 3CDJ, and 4CDJ also verified the OC, LIN, and SC DNA and household doenjang. Among them, 1CDJ demonstrated the strongest OC DNA band, followed by LIN and SC, and 2CDJ, 3CDJ, and 4CDJ exhibited the same DNA protective effect as a positive control.
Boukhris et al. [47] reported that flower extract caused no damage to DNA. Still, the bands' fluorescence intensity was altered because the bands of supercoil DNA weakened, and the open circle DNA became stronger when DNA damage occurred. Gao et al. [48] reported that the plasmid DNA is used to identify DNA damage as it is found in the form of supercoil. It was converted into a gentle open circular DNA and linear DNA form when H 2 O 2 and Fe 2+ were added. It also validated the highest DNA protective effect at a concentration of 200 mol/L of Shallercapus gracilis extract. Akher et al. [49] validated the high protecting activity of the supercoil DNA bands in extracts with ethanol and methanol of Boerhaavia diffusa, similar to the control. Additionally, it was reported that the DNA protective effect was low in samples where the supercoil DNA band's fluorescence intensity was reduced, and the circular DNA band's fluorescence intensity was increased. When plasmid DNA damage occurred, a phosphate ester bond of the supercoil DNA was cleaved to produce relaxed open circular DNA, and the portion near the DNA where the first crack occurred was cleaved and changed to linear double-stranded DNA [47,50,51]. In this regard, when the protecting activity against DNA damage is verified, the damage to supercoil DNA occurs the least. It was judged that the material with low open circular DNA and linear DNA formation has a high DNA protective effect. Therefore, 2HDJ, 3HDJ, 2CDJ, 3CDJ, and 4CDJ exhibited a similar DNA protective effect as the control without Fenton's reagent. Additionally, Marazza et al. [52] reported that as the soy milk fermented, DNA protective effect increased, and reported that the greatest DNA protective effect was shown at 24 h of fermentation. It was reported that this was because of the isoflavone aglycone contents, which varied from these results. In this regard, it was judged that it is necessary to verify the relationship with different compounds in addition to aglycones. These studies on the DNA protective effect of soybean and fermented foods using soybean for the Fenton reaction have been insignificant. Therefore, this study verifies the DNA protective effect of doenjang on the Fenton reaction and will be used as basic data for studying the DNA protection effect of soybean and soybean-fermented food.
produce relaxed open circular DNA, and the portion near the DNA where the first c occurred was cleaved and changed to linear double-stranded DNA [47,50,51]. In thi gard, when the protecting activity against DNA damage is verified, the damage to su coil DNA occurs the least. It was judged that the material with low open circular D and linear DNA formation has a high DNA protective effect. Therefore, 2HDJ, 3 2CDJ, 3CDJ, and 4CDJ exhibited a similar DNA protective effect as the control wit Fenton's reagent. Additionally, Marazza et al. [52] reported that as the soy milk fermen DNA protective effect increased, and reported that the greatest DNA protective effect shown at 24 h of fermentation. It was reported that this was because of the isofla aglycone contents, which varied from these results. In this regard, it was judged tha necessary to verify the relationship with different compounds in addition to aglyco These studies on the DNA protective effect of soybean and fermented foods using soy for the Fenton reaction have been insignificant. Therefore, this study verifies the D protective effect of doenjang on the Fenton reaction and will be used as basic data for s ying the DNA protection effect of soybean and soybean-fermented food.

Medium and Chemical
The LB broth and agar media were purchased from Difco (Becton Dickinson Spark, MD, USA). In order to measure the antioxidants and the enzyme activities, th

Collection of Doenjang Samples
The standard manufacturing process of household doenjang and commercial doenjang is shown in Figure 8. Traditional household doenjang is generally prepared at home in Gyeongnam province for a long time.

Collection of Doenjang Samples
The standard manufacturing process of household doenjang and commercial doenjang is shown in Figure 8. Traditional household doenjang is generally prepared at home in Gyeongnam province for a long time.

Determination of pH, Acidity, Salinity and Soluble Protein Content on Doenjang
The doenjang (10 g) was mixed with 90 mL distilled water and shaken for 30 min. Next, the supernatant was obtained by centrifugation (13,000× g, 3 min). The supernatant's pH was measured using a pH meter (MP 220 pH meter, Schwerzenbach, UK), and the acidity (lactic acid, %) was measured by titrating the supernatant with 0.1 NaOH until pH was 8.2 ± 0.1. For salinity measurements, 10 g doenjang was mixed with 40 mL distilled water, and the supernatant was obtained after shaking and centrifugation, as stated above. The supernatant's salinity was measured using a salt meter (PAL-03S, ATAGO, Japan).
The soluble protein contents were measured using the biuret method [53]. Four milliliters of biuret reagent and 1 mL doenjang sample extract were placed in a screw-capped test tube and kept in a hot water bath at 37 °C for 20 min. The absorbance was measured

Determination of pH, Acidity, Salinity and Soluble Protein Content on Doenjang
The doenjang (10 g) was mixed with 90 mL distilled water and shaken for 30 min. Next, the supernatant was obtained by centrifugation (13,000× g, 3 min). The supernatant's pH was measured using a pH meter (MP 220 pH meter, Schwerzenbach, UK), and the acidity (lactic acid, %) was measured by titrating the supernatant with 0.1 NaOH until pH was 8.2 ± 0.1. For salinity measurements, 10 g doenjang was mixed with 40 mL distilled water, and the supernatant was obtained after shaking and centrifugation, as stated above. The supernatant's salinity was measured using a salt meter (PAL-03S, ATAGO, Japan).
The soluble protein contents were measured using the biuret method [53]. Four milliliters of biuret reagent and 1 mL doenjang sample extract were placed in a screw-capped test tube and kept in a hot water bath at 37 • C for 20 min. The absorbance was measured at 540 nm using a spectrophotometer (Thermo Scientific GENESYS 20 Spectrophotometer, CA, USA). The crude protein concentration was measured using a bovine serum albumin standard curve.

Microbial Isolation and Identification
Eight doenjang samples were diluted with sterile physiological saline. The diluted samples (0.1 mL) were spread on tryptic soy agar plates, and incubated for 48 h at 37 • C, and after the generated colonies were measured. Each experiment was repeated three times as an average value and expressed as colony forming unit (log cfu/g) per gram of sample.
Selected colonies were cultivated in LB broth for 12 h with shaking (150 rpm) at 37 • C. The culture (3 mL) was centrifuged in sterile tube at 13,000× g for 3 min. Total DNA were isolated from the collected cells using G-spin genomic DNA Purification Kit (Intron Biotechnology, Suwon, Korea). The 16S rRNA gene sequences were confirmed as described by Cho et al. [45]. The determined 16S rRNA gene sequence was compared with another bacterial 16S rRNA obtained from the GeneBank database. The similarity values were calculated from alignments, evolutionary distances using a DNAMAN analysis system (Lynmon Biosoft, Quebec, Canada). Phylogenetic trees were identified using the neighbor-joining method and distance matrix data.

Preparation of Doenjang Extracts
One gram of freeze-dried powder sample was mixed with 50% methanol (20 mL) and shaken for 12 ± 2 h at room temperature. After that, the mixture was centrifuged, followed by extracting the supernatant with a syringe filter (diameter 25 mm, pore size 0.45 µm, Chromdisc, Maidstone, UK).

Determeination of the TP and TF Contents of Doenjang Extracts
The TP contents were determined using the Folin Denis method [54]. A total of 0.5 mL diluted extract was dispensed into a test tube, and a 0.5 mL 25% Na 2 CO 3 solution was added and kept for 3 min. Next, 0.25 mL of 2 N Folin Ciocalteu phenol reagent was added to the reaction solution and kept at 30 • C for 1 h. After that, the solution absorbance was measured at 750 nm. The total phenolic contents were measured as per the gallic acid standard curve.
The TF contents was determined by the method as described Lee et al. [55]. Briefly, 0.5 mL of diluted extract was dispensed into a test tube, and 1 mL of diethylene glycol and 0.01 mL of 1 N NaOH were added and reacted in a hot water bath at 37 • C for 1 h. The absorbance was measured at 420 nm using a spectrophotometer and the total flavonoid contents were measured as rutin standard curve.

DPPH Radical Scavenging Activity
The DPPH radical scavenging activity of each extract was determined using Lee et al. [54] method. In addition, 0.2 mL of each sample extract was mixed with 0.8 mL 1.5 × 10 −4 M DPPH reagent in ethanol. The mixture was incubated for 30 min at room temperature in the dark, and their absorbance was measured at 525 nm using a spectrophotometer. The extract solution was used as a negative control. The DPPH radical scavenging effects were calculated using the following equation:

ABTS Radical Scavenging Activity
The ABTS radical scavenging activity of each extract was measured according to Lee et al. [54]. The ABTS solution (a mixture of 5 mL 7 mM ABTS and 5 mL 2.45 mM K 2 S 2 O 8 ) was kept in the dark at room temperature for 12-16 h. After that, the solution was diluted using methanol to adjust its absorbance (OD) to around 0.7 ± 0.02 at 734 nm wavelength. Next, 0.1 mL each sample extracts were allowed to react with 0.9 mL ABTS solution for three minutes. Finally, the absorbance of the solutions was measured at a 732 nm wavelength in a spectrophotometer. The unreacted extract solution was used as a negative control. The ABTS radical scavenging effects were calculated using the following Equation (1).

Hydroxyl Radical Scavenging Activity
The hydroxyl radical scavenging activity of each doenjang extract was determined using Adjimani and Asare's [56] method. Briefly, 1.4 mL sample extracts were mixed with 0.2 mL 10 mM FeSO 4 .7H 2 O-EDTA, 0.2 mL 10 mM 2-deoxyribose, and 0.2 mL 10 mM H 2 O 2, consequently, the mixture was incubated at 37 • C for 4 h. Thenceforth, 1 mL 1% thiobarbituric acid and 1 mL 2.8% trichloroacetic acid were added to the reaction mixture and sunk in a hot water bath at 100 • C for 20 min. Next, the reactant mixture was cooled to room temperature to measure absorbance at 520 nm wavelength in a spectrophotometer. The phosphate buffer saline was used as a negative control during the absorbance measurement of the reactant samples. The hydroxyl radical scavenging effects were calculated using the following Equation (1).

Ferric Reducing/Antioxidant Power
The ferric reducing/antioxidant power (FRAP) of each doenjang extract was assayed as per the modified method of Wen et al. [57]. The FRAP reagent was mixed with acetate buffer (30 mM, pH 3.6), TPTZ reagent (10 mM in 40 mM HCl), and FeCl 3 solution (20 mM in DW) in a ratio of 10:1:1 (v/v/v). Next, the reaction mixture was allowed to react at 37 • C for 15 min in a water bath. Later, 0.95 mL FRAP reagent mixture was added with 0.05 mL doenjang extracts and incubated at 37 • C for 15 min. Finally, the solution absorbance was measured at 593 nm using a spectrophotometer.

DNA Damage Protecting Activities of Doenjang Extracts
DNA damage protecting activity of each doenjang extract was determined against SK+ vector plasmid DNA. Briefly, doenjang sample extracts, SK+ vector plasmid DNA, 1× TAE buffer (10 mM Tris-HCl and 1 mM EDTA), and Fenton's reagents (100 mM H 2 O 2 , 0.1 mM acetic acid and 1.6 mM FeCl 3 ) were prepared in advance. A negative control reaction mixture was made of 2 µL DNA (SK+ vector) and 18 µL sterile water. While the positive control reaction mixture comprised 2 µL DNA (SK+ vector), 10 µL doenjang sample extracts, and 8 µL Fenton's reagent. The experiment reaction mixture consisted of 2 µL DNA, 10 µL doenjang sample extract, and 8 µL Fenton's reagent. The reaction mixture was kept at 30 • C for 1 h in a water bath. After each reaction, the agarose gel (1.5%) electrophoresis was conducted to confirm the DNA degradation protection capabilities of the doenjang sample extracts [52].

Statistical Analyses of Nutritional Compositions and Antioxidant Properties
All determination values were based on three independent experiments with triplicate samples (n = 3). The results of nutritional component contents and antioxidant ratios in samples were represented as the mean ± SD values. Significant differences between samples were documented by Duncan's multiple range test (p < 0.05) through ANOVA procedure using the Statistical analysis system (SAS) software (ver. 9.4; SAS institute, Cary, NC, USA).

Conclusions
This study reported for the first time a variation in physicochemical features, antioxidant activities, secondary metabolites (TP, TF and isoflavone), cultivable bacterial diversities, and DNA protective effects of household and commercial doenjang. Characteristically, HDJ samples showed larger aglycone content ratios and greater diversity of bacterial distribution. In contrast, CDJ samples verified higher glycoside content ratios and relatively simple bacterial distribution. In particular, the greater ratios of bacterial strains were recorded as 49.9% B. subtilis in 1HDJ, 33.1% B. licheniformis in 2HDJ, 35.3% B. subtilis in 3HDJ, and 46.5% B. amyloliquefaciens in 4HDJ samples, while B. subtilis had the highest ratio 33.9-49.8% in common in CDJ samples. Therefore, it suggests that the greater portion of aglycone contents generation in HDJs were associated with bacterial diversity. However, the amounts of TP, TF, and isoflavone contents, antioxidant activities, and DNA protection effects were variedly observed regardless of HDJs and CDJs. These are estimated to be due to different manufacturing techniques included fermentation conditions, raw material content, soybean varieties, and cultivation environments (climate, temperature, and region). Due to enclosing higher aglycone contents ratios in HDJ samples, it is better to focus on how these biofunctional contents can be increased in CDJ production. Moreover, further studies should be conducted on the fermentation conditions, raw material, and enrichment of phytochemicals-in HDJs and CDJs.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author, upon reasonable request.